Overview: Integrin-initiated matrix adhesions play a central role in development and numerous pathological processes because they are essential for forming and shaping the extracellular matrix and for dictating the responses of cells to variations in matrix composition, stiffness, and to forces imposed on the matrix. The variability of these adhesions has led to ambiguous morphology-based classifications and thus hindered efforts to study their composition, structure, signaling properties, and relationship to biological functions such as protrusion morphodynamics. Adhesions dynamically evolve such that a continuum of intermediates is generally present. Our studies of the molecular connections of integrins and talins, the central organizers of matrix adhesions, have lead to the hypothesis that two mutually-exclusive binary protein-protein interactions underlie a central switch in the formation and maturation of integrin-based adhesions. Here we will test the idea that these protein-protein interactions can specify two distinct phenotypes of matrix-based adhesions, that these two adhesion types differ in composition, fine structure, signaling properties and consequences for protrusion morphodynamics and force sensing. Biological Significance of integrin-initiated Adhesions in Mechanotransduction. Integrin-based cell adhesions play central roles in the biology of metazeans(l) controlling cell adhesion to the extracellular matrix (ECM), migration, growth, differentiation and apoptosis In particular, these adhesions control both the assembly and rigidity of the extracellular matrix and the cell's capacity to sense rigidity(2-4). Similarly, signaling from these structures contributes to the ability of cells to exert and sense external fierce (5-7). In cells migrating in a mesenchymal mode, activated integrins associate with polymerizing actin at the leading edge and, in doing so, move within the plasma membrane, possibly seeking ECM binding sites(8). Nascent adhesions form at the cell anterior, and are generally smaller than the resolution of the light micrescepe(9). Some of the nascent adhesions disassemble within minutes(9), and the remainder grow and mature into focal complexes (-0.5 pm) and then FAs (1-5 pm);this evolution is driven by force(5, 10-13) and is thus a central feature of force-sensing by cells. There has been interest in defining the molecular composition, signaling properties, and ultimately the molecular structure of these adhesiens(14-18). A critical barrier to progress is the heterogeneity of these adhesions since, in any cell, a continuum of evolving forms is present, with marked variability in compositions, structures, and functions, Integrin Activation: A First Step in Adhesion Assembly. Most integrins are expressed in a low affinity state and require cellular activation to bind ECM with sufficient affinity to form adhesions. The binding of talins to the p subunit cytoplasmic tail is a final common step in integrin activation in wfro(19) and in vivo{20, 21) (22). In purified systems, talin binding is sufficient to activate integrins(23) and much has been learned about the details of talin-induced activation(24);in cells there is also a requirement for kindlins(25-30);however, the mechanism of kindlins'actions remains unknown. The spectrum of nascent to mature ECM adhesions contain talin(31) and talin is required for adhesion fermatien(32-34). Recent studies have begun to decipher how the talin-integrin interaction is regulated to control integrin activation? Ras GTPases are critical signaling modules in the activation process (35), The Ras subfamily members, Rapl a and Raplb, simulate integrin activation(36, 37) (38-40). Several Rapl effectors have been implicated in integrin activation(41-43), RIAM (Rapl-GTP-interacting adapter molecule) is a Rapl effector that is a member of the MRL (Mig-10/RIAM/Lpd) family of adaptor proteins(42). RIAM contains Ras association (RA) and pleckstrin homology (PH) domains and proline-rich regions, which are defining features ofthe MRL protein family. RIAM over expression induces pi and 32 integrin-mediated cell adhesion, and RIAM knockdown inhibits Rap1-dependent leukocyte adhesien(42), indicating RIAM is a downstream regulator of Rap1 dependent signaling. Whereas RIAM is greatly enriched in hematopoietic cells, Lpd (Lpd) is a paralogue also present in fibroblasts and ether somatic cells(44). We exploited the fact that agonists fail to activate recombinant allbB3 expressed in CHO cells to develop a synthetic reconstruction of an integrin activation pathway and used it in combination with forward and reverse genetics to dissect a pathway to integrin activation(45). We found that Rap1-induced formation of an integrin activation complex containing the Rap1 effector, RIAM, and talin that lead to talin recruitment to the plasma membrane and to integrin allbB3. More recently, we further reconstructed this pathway to demonstrate activation of allbB3 by stimulation of a physiologically relevant receptor, PARI, and used bimolecular fluorescence complementation to show that RIAM over expression stimulates and RIAM knockdown blocks talin recruitment to allbB3 in living cells(46). Furthermore, mapping studies identified short amphipathic helices in RIAM and Lpd that bind talin;joining those helical peptides to the membrane targeting sequences of Rap1 led to a minimized Rap-RIAM module that was sufficient to recruit talin to integrins and to activate the integrins(47). Thus, RIAM functions as a scaffold that connects the membrane-targeting sequences in Ras GTPases to talin, thereby recruiting talin to the plasma membrane and activating integrins. Together, these results enable us to construct a skeletal "Roadmap" to initial integrin activation (Fig.1).